HIV-1-specific CD8+ T cells are associated with declining viremia in acute primary infection and control of viral replication in long-term non-progressive infection, but neither their magnitude nor breadth correlates with control of viremia in chronic progressive infection, suggesting a functional defect of these cells that is not measured by interferon-g secretion assays commonly used. In addition, a series of previous studies indicated that the antigen-specific secretion of TNF-a and MIP-1b or antigen-specific degranulation in HIV-1- specific CD8+ T cells are also not correlated with protective immunity against HIV-1. The definition of functional properties of HIV-1-specific CD8+ T cells that are associated with protection against HIV-1 disease progression is therefore of primary importance for the design of HIV-1 immunogens. Here, we propose to use a novel functional proteomics approach to analyze HIV-1-specific CD8+ T cells on the level of post-TCR signaling. Our overall hypothesis is that the analysis of TCR-triggered phosphorylation of signal transduction proteins (kinomics) allows for identifying HIV-1-specific CD8+ T cells associated with a protective CD8+ T cell response against HIV-1. To test this hypothesis, we will compare TCR signaling between HLA-A2- restricted HIV-specific CD8+ T cells and A2-restricted CD8+ T cells specific for immunologically-controlled viruses, such as CMV, EBV and Influenza virus, using a high-throughput multiplex reverse-phase protein microarray for screening purposes and flow cytometric assays with antibodies recognizing phosphorylated kinases for the in-depth analysis of signaling events on a single cell level (specific aim #1a). Moreover, we will analyze whether TCR signaling pathways differ between HI V-1-specific CD8+ T cells restricted by HLA- class I alleles associated with distinct HIV-1 disease outcomes (specific aim #1b). Finally, we will test whether specific clusters of post-TCR signaling pathways are associated with the execution of selected effector functions of HI V-1-specific CD8+ T cells (specific aim #2), again using microarray screening technology in combination with flow cytometry. These experiments will indicate whether specific functional characteristics of HI V-1-specific CD8+ T cells, such as cytokine secretion or cytotoxic properties, are associated with distinct signaling characteristics. Overall, our investigations will be relevant for defining key signaling networks of HI V-1-specific CD8+ T cells associated with protective immunity against HIV-1. [unreadable] [unreadable]